Project Summary/Abstract Alkyl DNA adducts are cytotoxic and mutagenic DNA lesions that arise from exposure of cells to numerous environmental carcinogens and cellular metabolites. Some of the most toxic alkyl DNA adducts are induced by commonly used anti-cancer drugs. Simple alkylating agents such as methyl methanesulfonate (MMS) and the chemotherapy drug Temozolomide (TMZ) induce a spectrum of highly cytotoxic and mutagenic methyl DNA adducts: N3-methyladenine (3meA), N7-methylguanine (7meG) and O6-methylguanine (O6meG). To better understand how alkyl DNA adducts contribute to cancer development and treatment, it is essential to determine the distribution of adducts across the genome and assess how factors (such as the genomic and epigenomic landscape of the cell) influence adduct formation and repair. Existing methods used to map and quantify alkyl DNA adducts in genomic DNA do not allow high throughput, high resolution, genome-wide mapping of multiple types of adducts. The primary objective of this proposal is to develop a novel method for direct detection of alkyl DNA adducts (ADA) using single molecule real time (SMRT) DNA sequencing. High resolution mapping of various alkyl DNA adducts by ADA-SMRT will enable comprehensive, high throughput characterization of these lesions across eukaryotic genomes. Implementation of ADA-SMRT will entail determining the SMRT kinetic signatures for DNA adducts induced by alkylating agents, and then mapping the pattern of distribution of the adducts across the yeast genome following MMS exposure (Aim 1). We will also develop a complementary MDA-seq approach to provide genome-wide profiling of two of the common alkylation-induced lesions (N-methyl purines, 3meA and 7meG) and provide validation to ADA-SMRT (Aim 2). Using ADA-SMRT (and/or MDA-seq), we will analyze formation and repair of MMS-induced alkyl DNA adducts in the genomes of wild-type and repair-deficient yeast to gain insights into how the heterogenous landscape of the genome and epigenome affect the distribution and repair of alkyl DNA adducts (Aim 3). The goal of these studies is to uncover novel patterns and/or unique hot spots of alkyl DNA adducts that influence genome stability, mutagenesis, and carcinogenesis. Importantly, development and implementation of ADA-SMRT and MDA-seq technology will allow assessment of numerous factors in alkyl-induced lesion formation and repair, and carcinogenesis. For example, the methodology developed in this work will provide a framework for future investigation and understanding of the individual differences in cancer predisposition and response to chemotherapy. This methodology could also be for detection of alkyl DNA adducts arising from endogenous, environmental or chemotherapy-associated sources in human biospecimens and tissues. In summary, the proposed new technology is expected to have a major impact on understanding the genomic and epigenomic basis of cancers induced by alkylating agents and will facilitate development of new approaches in cancer risk identification, prevention and personalized cancer treatment.